Cd4 t cell microbeads

Cell-associated HIV-1 DNA was isolated from purified CD4⁺ T cells (CD4⁺ T cell microbeads, Miltenyi) from final quantities of 2x10⁶ cells per patient. Extraction was done using the DNeasy Blood and tissue kit (Qiagen, Hilden, Germany) according to manufacturer’s instruction. Absolute quantification of HIV-1 DNA was performed by droplet digital PCR (ddPCR) (Bio-Rad, Hercules, California) as previously described (35 (link)) with total HIV-1 DNA primers and probes as reported (36 (link)).

Splenic CD4⁺T cells were isolated from wild‐type mice (6‐8 weeks old) using CD4⁺T‐cell microbeads (Miltenyi Biotec). For Th17 cell differentiation,10 CD4⁺T cells were cultured for 72 hours in the presence of anti‐CD28 mAb (2 μg/mL), IL‐6 (30 ng/mL), TGF‐β(5 ng/mL), anti‐IFN‐γmAb (5 μg/mL), anti‐IL‐4 mAb (5 μg/mL) and IL‐23 (30 ng/mL) in a 24‐well plate pre‐coated with anti‐CD3 mAb (2 μg/mL).
EC‐derived exosomes regulate Th17 cell differentiation in vitro were assigned to the following five groups: (a) control; (b) con‐Exo; (c) CD137‐Exo; (d) si IL‐6; and (e) si‐NC.

To perform the co-culture experiments for a functional assay of T cell proliferation, CD4⁺ T cells were isolated from PBMCs using the CD4⁺ T cell microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions and sorted on an autoMACS classic (Miltenyi Biotec, Bergisch-Gladbach, Germany) using the program ‘Possel’. The autologous CD4⁺ T cells were stained for CFSE and rested in complete RPMI for 24 h at 37^o C and 5% CO₂.
Monocytes were left unstimulated or stimulated with 20 μg/ml of Mf or cystatin-depleted Mf lysate for 24 h in a 5% CO₂ incubator at 37^o C in 24 well plates. 5x10⁵ CFSE-labelled CD4⁺ T cells were co-cultured with 1x10⁵ stimulated monocytes in 96 well flat bottom plates in the presence of 2 μg/ml soluble anti-CD3 (OKT3, eBioscience, San Diego, USA). After 5 days, the cells were washed and stained with fixable viability dye eFluor 780 (eBioscience, San Diego, USA) and anti-CD4-PerCP (clone RPA-T4, BioLegend, San Diego, USA). Fixed cells were acquired using the FACS Canto II (BD, Franklin Lakes, USA) and analysed using FlowJo, version 10.2 (Tree Star, Ashland USA).